Methods and Compositions for Treating Diabetes

ABSTRACT

Described herein are methods and compositions for treating diabetes mellitus, concerning oral pharmaceutical compositions comprising insulin in combination with a GLP-1 analogue.

Benefit is claimed to U.S. Provisional Application 61/631,339, filedJan. 3, 2012, which is incorporated herein by reference in its entirety.

FIELD

Described herein are methods and compositions for treating diabetesmellitus.

BACKGROUND

Diabetes, specifically the Type II (NIDDM) variety, has emerged in thetwenty-first century as an epidemic of global proportions. Numerouslong-term complications, including those affecting the kidneys, legs,feet, eyes, heart, nerves, and blood circulation, may result fromuncontrolled diabetes. Prevention of these conditions requirescomprehensive treatment, requiring life style modification andmedication. A number of effective anti-diabetic drugs are available andare generally safe and well tolerated. All the medications become lesseffective as the disease progresses, and most patients eventuallyrequire insulin. Most of the medications are associated with risks ofhypoglycemia and weight gain, yet do not alter the inexorableprogression of diabetes.

Orally-delivered formulations for protein-based drugs such as insulinare being developed by the present inventor (Ziv et al 1994; Nissan etal 2000, Kidron et al 2004, Eldor et al 2010B, Eldor et al 2010C). Onesuch oral insulin product is scheduled to be tested in Phase II trialsand is currently being reviewed for IND status.

The incretin hormone Glucagon-like Peptide 1 (GLP-1), secreted withinminutes of food ingestion, is associated with induction of insulinrelease. Therapies based on GLP-1 are treatment options for Type 2Diabetes Mellitus (T2DM) that act through a variety of complementarymechanisms. The most intriguing aspect of the incretins is the fact thatthey cause insulin release in a glucose-dependent manner and are thoughtto have a low risk of inducing hypoglycemia. Furthermore, the incretinsseem to be weight-neutral (or weight-reducing), preserve beta-cell mass,and possibly also induce neogenesis of insulin-secreting cells.

However, clinical use of the native GLP-1 is limited due to its rapidenzymatic inactivation, resulting in a half-life of 2-3 minutes. Toovercome this obstacle, long-acting degradation-resistant peptides, bothnatural and synthetic, referred to as GLP-1 mimetic agents or analogues,have been designed and tested.

To date, GLP-1 analogues are only available as injectable dosage forms.The present inventor is developing an oral exenatide GLP-1 analoguecapsule. A first-in-humans trial (n=4) testing its safety in healthyhumans demonstrated retained biological functionality of orallydelivered exenatide (Eldor et al 2010A).

SUMMARY

To the inventor's knowledge, oral insulin formulations have not beentested in combination with oral GLP-1 analogue formulations. The dataprovided herein illustrate a previously-unrecognized, strong cooperativeinteraction between these components when formulated as describedherein. This enables a potent anti-diabetes effect in a convenient formthat both facilitates patent compliance and also mimics physiologicalfirst-pass metabolism of insulin and GLP-1. These results provide aroute to an entirely new class of therapeutic modalities.

The terms “protein” and “peptide” are used interchangeably herein.Neither term is intended to confer a limitation of the number of aminoacids present, except where a limitation is explicitly indicated.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are by way of illustrative example and are notmeant to be taken as limiting the claimed invention.

FIG. 1. Testing of various emulsifier formulations. Foam buildup scorewas from 1-5, where 1 indicates no foam, and 5 indicates no liquidvisible because of the foam. For the suspension test, the numbers 1-5indicate full phase separation; partial phase separation with somelarger oil bubbles; small oil bubble, milky consistency; no bubblesinitially, with later phase separation; and stable emulsion,respectively.

FIGS. 2A and 2B. Blood glucose profiles following administration of oralinsulin formulations containing various emulsifiers. 2A: Formulations A(upper left), B (lower left), C (upper right), and D (lower right). 2B:Formulations E (left) and F (right).

FIG. 3. Blood glucose profiles following oral exenatide and oral insulinadministration to pigs. Fasting, commercial pigs were treated with 150mcg. exenatide, 8 mg. insulin, or the combination thereof, 30 minutesbefore caloric intake. 1-mL blood samples were periodically drawnthroughout the 180-minute observation period to determine glucoseconcentrations.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Provided herein is a pharmaceutical composition for oral delivery,comprising an oil-based liquid formulation, the oil-based liquidformulation comprising an insulin, a GLP-1 analogue, a trypsininhibitor, and a chelator of divalent cations, wherein the oil-basedliquid formulation is surrounded by a coating or capsule that resistsdegradation in the stomach.

Another embodiment provides a multi-component oral pharmaceuticalcomposition, comprising: (a) a first oil-based liquid formulation, thefirst oil-based liquid formulation comprising an insulin, a trypsininhibitor, and a chelator of divalent cations; and (b) a secondoil-based liquid formulation, the second oil-based liquid formulationcomprising a GLP-1 analogue, a trypsin inhibitor, and a chelator ofdivalent cations; wherein each of the first oil-based liquid formulationand the second oil-based liquid formulation is surrounded by a coatingor capsule that resists degradation in the stomach. In some embodiments,the two liquid formulations can be in separate dosage forms. In otherembodiments, the two liquid formulations are in the same dosage form;for example, in separate encased compartments within the same pill.

“Liquid” as used herein refers to a phase that flows freely and has aconstant volume under ambient conditions. Fish oil, for instance, is aliquid under ambient conditions. The term includes oil-based solutions,suspensions, and combinations thereof. In alternative embodiments, theterm may refers to a composition that has a viscosity within the rangeof 1-1000 millipascal seconds, inclusive, at 20° C.

In certain embodiments, the different components of a multi-componentpharmaceutical composition are indicated for co-administration together.“Co-administration”, in this regard, may refer either to simultaneousadministration or, in another embodiment, to administration within 30minutes of each other. In still other embodiments, the differentcomponents are indicated for administration in a particular order,separated by a set time interval that will typically be 30 minutes orless. For example, the insulin-containing dosage form may be indicatedfor administration 2-10 minutes after the exenatide-containing dosageform; in other embodiments, 10-20 minutes after the exenatide-containingdosage form; in other embodiments, 20-30 minutes after theexenatide-containing dosage form; and in other embodiments, 30-60minutes after the exenatide-containing dosage form. Oral dosage formssuch as those provided herein lend themselves to sequentialadministration more than injected dosage forms, since regimens requiringrepeated injections are likely to be associated with low rates ofcompliance.

According to other aspects, a combination medicament for treatment oftype 2 diabetes is provided, said combination medicament comprising

-   -   insulin, at least one trypsin inhibitor, and a chelator of        divalent cations, and    -   a GLP-1 analogue from the group consisting of exenatide,        liraglutide, AC3174, taspoglutide, lixisenatide, semaglutide,        albiglutide, exendin-9, LY2189265, and CJC-1134-PC,    -   all comprised jointly in an oil-based liquid formulation in a        dosage form for oral delivery.

Insulin Proteins and GLP-1 Analogues

Insulin proteins and GLP-1 analogues for use as described herein are insome embodiments isolated prior to their introduction into the describedpharmaceutical compositions. “Isolated” in this regard excludesprovision of the insulin and/or GLP-1 analogue as a homogenized tissuepreparation or other form containing substantial amounts ofcontaminating proteins. An example of an isolated protein or peptide isa recombinant protein or peptide. An alternative embodiment is asynthetic protein or peptide.

A person skilled in the art will appreciate in light of the presentdisclosure that various types of insulin are suitable for the describedmethods and compositions. Exemplary insulin proteins include but are notlimited to both wild-type and mutated insulin proteins, includingsynthetic human insulin, synthetic bovine insulin, synthetic porcineinsulin, synthetic whale insulin, and metal complexes of insulin, suchas zinc complexes of insulin, protamine zinc insulin, and globin zinc.

Various classes of insulin may also be utilized, for example fast-actinginsulin, lente insulin, semilente insulin, ultralente insulin, NPHinsulin, glargine insulin, lispro insulin, aspart insulin, orcombinations of two or more of the above types of insulin.

In certain embodiments, the insulin of the described methods andcompositions is wild-type human insulin (Uniprot ID P01308). In someembodiments, human insulin is produced as a recombinant protein inbacterial cells. In other embodiments, human insulin is producedsynthetically.

GLP-1 analogues are also referred to in the art as GLP-1 mimetics. Aperson of skill in the art will appreciate that the describedcompositions may include at least one of the following GLP-1 analogues:exenatide (Byetta™; CAS no. 141732-76-5; SEQ ID NO: 4), lixisenatide(CAS no. 320367-13-3), liraglutide (CAS no. 204656-20-2), exendin-9 (CASno. 133514-43-9), AC3174 ([Leu(14)]exendin-4, Amylin Pharmaceuticals,Inc.), taspoglutide (CAS no. 275371-94-3), albiglutide (CAS no.782500-75-8), semaglutide (CAS no. 910463-68-2), LY2189265(Dulaglutide™; CAS no. 923950-08-7), and CJC-1134-PC (a modifiedExendin-4 analogue conjugated to recombinant human albumin manufacturedby ConjuChem™). All CAS records were accessed on Dec. 19, 2011. Thus, incertain embodiments, the described method or composition utilizes any ofthe above-listed GLP-1 analogues. In other embodiments, one of theabove-listed GLP-1 analogues is selected. Those of skill in the art willappreciate in light of the findings of described herein that other GLP-1analogues can also be utilized.

Therapeutic insulin and GLP-1 proteins suitable for use in the presentinvention include derivatives that are modified (i.e., by the covalentattachment of a non-amino acid residue to the protein). For example, butnot by way of limitation, the protein includes proteins that have beenmodified, e.g., by glycosylation, acetylation, PEGylation,phosphorylation, amidation, or derivatization by knownprotecting/blocking groups. High-MW PEG can be attached to therapeuticproteins with or without a multifunctional linker either throughsite-specific conjugation of the PEG to the N- or C-terminus thereof orvia epsilon-amino groups present on lysine residues. Additionally, thederivative may contain one or more non-classical amino acids, forexample D-isomers of the common amino acids, 2,4-diaminobutyric acid,α-amino isobutyric acid, A-aminobutyric acid, Abu, 2-amino butyric acid,γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Ca-methyl amino acids, and Nα-methyl amino acids.

Emulsifiers

In certain embodiments, an oil-based liquid formulation utilized in thedescribed methods and pharmaceutical compositions, or in otherembodiments, each of the oil-based liquid formulation that is present,further comprises a component provided as a mixture of (a) amonoacylglycerol (monoglyceride), a diacylglycerol (diglyceride), atriacylglycerol (triglyceride), or a mixture thereof; and (b) apolyethylene glycol (PEG) ester of a fatty acid. In this regard, each ofthe terms “monoacylglycerol”, “diacylglycerol”, and “triacylglycerol”need not refer to a single compound, but rather can include mixtures ofcompounds, for example mixtures of monoacylglycerols, diacylglycerols,or triacylglycerols having fatty acids of varying lengths. In certainpreferred embodiments, monoacylglycerols, diacylglycerols, ortriacylglyccrols utilized in the described methods and compositions, forexample those used to general PEG esters, are from an oil source that isGenerally Recognized As Safe (GRAS). Examples of GRAS oil sources arecoconut oil, corn oil, peanut oil, soybean oil, Myvacet 9-45(Diacetylated monoglycerides of C-18 fatty acids).

A more specific embodiment of (a) is a mixture of C₈-C₁₈monoacylglycerols, diacylglycerols, and triacylglycerols. A morespecific embodiment of component (b) is a mixture of PEG monoesters anddiesters of a mixture of C₈-C₁₈ fatty acids.

In other, more specific embodiments, the liquid formulation furthercomprises a free PEG.

In alternate embodiments, an oil-based liquid formulation utilized inthe described methods and pharmaceutical compositions, or in otherembodiments, each of the oil-based liquid formulation that is present,further comprises a PEG ester of a monoacylglycerol, a diacylglycerol, atriacylglycerol, or a mixture thereof. In this regard, each of the terms“monoacylglycerol”, “diacylglycerol”, and “triacylglycerol” need notrefer to a single compound, but rather can include mixtures ofcompounds, for example mixtures of monoacylglycerols, diacylglycerols,or triacylglycerols having fatty acids of varying lengths. In morespecific embodiments, an additional non-ionic detergent, for example apolysorbate-based detergent, is present in addition to the PEG ester. Inother, more specific embodiments, a free PEG is also present. In stillmore specific embodiments, both an additional non-ionic detergent and afree PEG are also present.

In a still more specific embodiment of the described methods andcompositions, a liquid formulation used therein comprises: (a) a mixtureof C₈-C₁₈ monoacylglycerols, diacylglycerols, and triacylglycerols; (b)PEG-32 monoesters and diesters of a mixture of C₈-C₁₈ fatty acids; and(c) free PEG-32. Even more specifically, the weight/weight ratio ofcomponent (a) to components (b)+(c) is between 10:90-30:70 inclusive;more specifically between 15:85-25:75 inclusive; more specifically20:80. In certain embodiments, components (a)-(c) together constitute8-16% weight/weight inclusive of the oil-based liquid formulation. Inmore specific embodiments, the amount is 9-15% inclusive. In morespecific embodiments, the amount is 10-14% inclusive. In more specificembodiments, the amount is 11-13% inclusive. In more specificembodiments, the amount is 12%.

In other embodiments, an oil-based liquid formulation utilized in thedescribed methods and pharmaceutical compositions further comprises aself-emulsifying component, which may or may not be one of the mixturesof components described in the preceding paragraphs. “Self-emulsifyingcomponent” in some embodiments refers to a component that spontaneouslyforms an emulsion. Typically, such components will form an emulsionunder on contact with aqueous media, forming a fine dispersion i.e. amicroemulsion (SMEDDS). Certain embodiments of such components comprisea mixture of triacylglycerols and a high hydrophile/lipophile balance(HLB; see Griffin W C: “Calculation of HLB Values of Non-IonicSurfactants,” J Soc Cosmetic Chemists 5:259 (1954)) surfactant. Otherembodiments of the self-emulsifying component have a waxy, semi-solidconsistency.

Preferably, the HLB of a self-emulsifying component utilized in thedescribed methods and compositions is 10 or greater. In otherembodiments, it is between 11-19 inclusive. In other embodiments, it isbetween 12-18 inclusive. In other embodiments, it is between 12-17inclusive. In other embodiments, it is between 12-16 inclusive, which isindicative of an oil-in-water (O/W) emulsifier. In other embodiments, itis between 13-15 inclusive. In other embodiments, it is 14. Still morespecific embodiments of self-emulsifying components have an HLB of 12-16inclusive and comprise medium and long chain triacylglycerols conjugatedto PEG, free triacylglycerols, and free PEG. In other embodiments, theself-emulsifying component has an HLB of 12-16 inclusive and consists ofa mixture of medium and long chain triacylglycerols conjugated to PEG,free triacylglycerols, and free PEG. In other embodiments, theself-emulsifying component has an HLB of 14 and comprises medium andlong chain triacylglycerols conjugated to PEG, free triacylglycerols,and free PEG. In other embodiments, the self-emulsifying component hasan HLB of 14 and consists of a mixture of medium and long chaintriacylglycerols conjugated to PEG, free triacylglycerols, and free PEG.

Certain, more specific embodiments utilize self-emulsifying componentsthat comprise (a) a monoacylglycerol, a diacylglycerol, atriacylglycerol, or a mixture thereof; and (b) a polyethylene glycol(PEG) ester of a fatty acid. In this regard, each of the terms“monoacylglycerol”, “diacylglycerol”, and “triacylglycerol” need notrefer to a single compound, but rather can include mixtures ofcompounds, for example mixtures of monoacylglycerols, diacylglycerols,or triacylglycerols having fatty acids of varying lengths. A morespecific embodiment is a mixture of C₈-C₁₈ monoacylglycerols,diacylglycerols, and triacylglycerols.

A more specific embodiment of component (b) is a mixture of PEGmonoesters and diesters of a mixture of C₈-C₁₃ fatty acids.

In other, more specific embodiments, the self-emulsifying componentfurther comprises molecules of free PEG.

Preferred lengths of PEG moieties for use in the described compositionsand methods contain between 5-100 monomers. In more specificembodiments, the PEG may contain between 15-50 monomers. In still morespecific embodiments, the PEG may contain between 25-40 monomers. Inmore specific embodiments, the PEG may contain 32 monomers.

In a still more specific embodiment of the described methods andcompositions, a self-emulsifying component used therein comprises: (a) amixture of C₈-C₁₈ monoacylglycerols, diacylglycerols, andtriacylglycerols; (b) PEG-32 monoesters and diesters of a mixture ofC₈-C₁₈ fatty acids; and (c) free PEG-32; and the weight/weight ratio ofcomponent (a) to components (b)+(c) is 20:80. In certain embodiments,such a component constitutes 8-16% weight/weight inclusive of theoil-based liquid formulation. In more specific embodiments, the amountis 9-15% inclusive. In more specific embodiments, the amount is 10-14%inclusive. In more specific embodiments, the amount is 11-13% inclusive.In more specific embodiments, the amount is 12%.

Examples of self-emulsifying components meeting the above specificationsare Gelucire™ 44/14, Gelucire™ 53/10, and Gelucire™ 50/13. Aparticularly preferred example is Gelucire™ 44/14. The suffixes 44 and14 refer respectively to its melting point and itshydrophilic/lypophilic balance (HLB). Gelucire™ 44/14 (Gattefossé SAS,Saint-Priest, France) is obtained by polyglycolysis of hydrogenatedcoconut oil (medium and long chain triacylglycerols with PEG-32. It hasa hydrophile/lipophile balance of 14. It is composed of a definedadmixture of C₈-C₁₈ mono-, di- and triacylglycerols (20% w/w); PEG-32mono- and diesters and free PEG-32 (80% w/w). The main fatty acidpresent is lauric acid, accounting for 45% on average of the total fattyacid content. It is a solid dispersion composed of a PEG ester fractionunder a lamellar phase of 120 Å with a helical conformation and anacylglycerol fraction under a hexagonal packing. The main products ofsimulated gastrointestinal lipolysis of Gelucire™ 44/14 are PEG-32 monoand diesters.

Non-Ionic Detergents

In certain embodiments, the oil-based liquid formulation utilized in thedescribed methods and pharmaceutical compositions further comprises anon-ionic detergent in addition to the self-emulsifying component. Incertain embodiments, the non-ionic detergent is selected from the groupconsisting of polysorbate-20, polysorbate-40, polysorbate-80,lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenatedcastor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65and 80, sucrose fatty acid ester, methyl cellulose, carboxymethylcellulose, n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton™-X-100,Triton™-X-114, Thesit™, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), and N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate. Inother embodiments, one of the above-listed non-ionic detergents isselected.

In certain, more specific embodiments, a non-ionic detergent used in thedescribed methods and compositions is a polysorbate-based detergent.Examples of polysorbate-based detergent are detergents derived bycovalently bonding polyethoxylated sorbitan to a fatty acid. Morespecific embodiments of polysorbate-based detergents are polysorbate-20,polysorbate-40, and polysorbate-80.

For example, polysorbate 80 (Tween-80) is a mild, non-ionic detergentderived from polyethoxylated sorbitan and oleic acid and having thefollowing structure:

In the case of polysorbate 80, the moiety shown on the right side is amixture of fatty acids, containing 60-70% oleic acid (as depicted), withthe balance being primarily linoleic, palmitic, and stearic acids.

In a more specific embodiment, the polysorbate 80 constitutes 3-10%weight/weight inclusive of an oil-based liquid formulation used in thedescribed methods and compositions. In a more specific embodiment, thepercentage is 4-8% inclusive. In a more specific embodiment, thepercentage is 4.5-6% inclusive. In a more specific embodiment, thepercentage is 5%.

Dosages

Alternatively or in addition, the insulin and/or GLP-1 analogue presentin the described compositions or used in the described methods ispresent in a subclinical amount. The term “subclinical amount” in thiscontext refers to an amount less than that required to elicit a completedesired physiological effect, for example control of post-prandial bloodglucose levels, in the context of its formulation and the patient.Accordingly, a subclinical dose of insulin would be less than thatrequired using formulations similar to those described herein thatcontain insulin but lack a GLP-1 analogue; or, in a more specificembodiment, a formulation identical except for the absence of the GLP-1analogue. Similarly, a subclinical dose of a GLP-1 analogue would beless than that required using formulations similar to those describedherein that contain a GLP-1 analogue but lack insulin; or, in a morespecific embodiment, a formulation identical except for the absence ofthe insulin.

Those skilled in the art will appreciate, in light of the presentdisclosure, that characterization of a dose as subclinical will dependon the weight and health status (including insulin resistance, ifrelevant) of the patient, the circumstances of the administration,co-administration of other diabetes medications, the robustness of theactive ingredient and the excipients, and the desired physiologicaleffect. For example, studies to date of oral formulations similar tothose described herein, but containing insulin only, have shown that 8mg of an encapsulated oral formulation in combination with a proteaseinhibitor and EDTA, in fish oil (similar to the one described herein butlacking exenatide) is a subclinical dose for fasting, adult, human Type2 diabetic patients, if the goal is a robust change in blood glucoselevels; while 16 mg. is a clinical dose under the same circumstances.Doses of the same formulation necessary to achieve modulation ofpost-prandial glucose excursions in the same patients have not beendetermined, but are likely to be slightly higher. However, doses such asthese also depend on the potency of the formulation, and thus theclinical dose threshold may be slightly lower if a more potent proteaseinhibitor is used, for example. Determination of a subclinical dose fora particular set of circumstances, for example via empirical testing, iswell within the ability of one skilled in the art.

In more specific embodiments, the subclinical amount of insulin of thedescribed methods and compositions is between 6-16 mg inclusive for anadult patent having diabetes mellitus, for example Type 2 diabetesmellitus (T2DM), for example for preventing post-prandial glucoseexcursions when administered between 30-60 minutes (min) before a meal,in more specific embodiments 30 min, 45 min, or 60 min before a meal. Inother embodiments, the subclinical amount is between 6-14 mg inclusive.In other embodiments, the subclinical amount is between 6-12 mginclusive. In other embodiments, the subclinical amount is between 6-10mg inclusive. In other embodiments, the subclinical amount is 8 mg. Inother embodiments, the subclinical amount is 12 mg. In otherembodiments, the subclinical amount is between 8-16 mg inclusive. Inother embodiments, the subclinical amount is between 8-14 mg inclusive.In other embodiments, the subclinical amount is between 8-12 mginclusive. In other embodiments, the subclinical amount is between 8-10mg inclusive. In other embodiments, the subclinical amount is 16 mg. Inother embodiments, the subclinical amount is between 10-16 mg inclusive.In other embodiments, the subclinical amount is between 10-14 mginclusive. In other embodiments, the subclinical amount is between 10-18mg inclusive.

In other embodiments, the subclinical amount of insulin of the describedmethods and compositions is between 0.06-0.16 mg/kg (milligrams perkilogram body weight) inclusive for an adult patent having T2DM, forexample for preventing post-prandial glucose excursions whenadministered before a meal. In other embodiments, the subclinical amountis between 0.06-0.14 mg/kg inclusive. In other embodiments, thesubclinical amount is between 0.06-0.12 mg/kg inclusive. In otherembodiments, the subclinical amount is between 0.06-0.10 mg/kginclusive. In other embodiments, the subclinical amount is 0.08 mg/kg.In other embodiments, the subclinical amount is 0.12 mg/kg. In otherembodiments, the subclinical amount is 0.16 mg/kg. In other embodiments,the subclinical amount is between 0.08-0.16 mg/kg inclusive. In otherembodiments, the subclinical amount is between 0.08-0.14 mg/kginclusive. In other embodiments, the subclinical amount is between0.08-0.12 mg/kg inclusive. In other embodiments, the subclinical amountis between 0.08-0.10 mg/kg inclusive. In other embodiments, thesubclinical amount is between 0.10-0.16 mg/kg inclusive. In otherembodiments, the subclinical amount is between 0.10-0.18 mg/kginclusive. In other embodiments, the subclinical amount is between0.10-0.14 mg/kg inclusive.

In still other embodiments, the subclinical amount of insulin is anamount corresponding to one of the above amounts or ranges for an adult,adjusted per body weight for a pediatric patient. In other embodiments,the insulin is present in a subclinical amount adjusted for a pediatricpatient, and the GLP-1 analogue is also present in a subclinical amountadjusted for a pediatric patient.

The above amounts may be for wild-type human insulin, or in anotherembodiment, for one of the other types of insulin known in the art.

In other, more specific embodiments, a subclinical amount of a GLP-1analogue is present in a dosage form of the described methods andcompositions. In some embodiments, 150 micrograms (mcg), 200 mcg, 250mcg, or 300 mcg is considered a subclinical dose for adult, humansubjects with T2DM for example for preventing post-prandial glucoseexcursions when administered between 30-60 min before a meal, in morespecific embodiments 30 min, 45 min, or 60 min before a meal. In otherembodiments, the subclinical amount of GLP-1 analogue is between 100-400mcg inclusive for an adult patent having T2DM. In other embodiments, thesubclinical amount is between 100-300 mcg. inclusive. In otherembodiments, the subclinical amount is between 100-250 mcg. inclusive.In other embodiments, the subclinical amount is between 100-200 mcg.inclusive. In other embodiments, the subclinical amount is between100-150 mcg. inclusive. In other embodiments, the subclinical amount is100 mcg. In other embodiments, the subclinical amount is 150 mcg. Inother embodiments, the subclinical amount is 200 mcg. In otherembodiments, the subclinical amount is 250 mcg. In other embodiments,the subclinical amount is 300 mcg. In other embodiments, the subclinicalamount is between 150-400 mcg. In other embodiments, the subclinicalamount is between 150-300 mcg. inclusive. In other embodiments, thesubclinical amount is between 150-250 mcg. inclusive. In otherembodiments, the subclinical amount is between 150-200 mcg. inclusive.

In other embodiments, the subclinical amount of GLP-1 analogue of thedescribed methods and compositions is between 0.100-0.400 mcg/kginclusive for an adult patent having T2D, for example for preventingpost-prandial glucose excursions when administered before a meal. Inother embodiments, the subclinical amount is between 0.100-0.300 mcg/kginclusive. In other embodiments, the subclinical amount is between0.100-0.250 mcg/kg inclusive. In other embodiments, the subclinicalamount is between 0.100-0.200 mcg/kg inclusive. In other embodiments,the subclinical amount is between 0.100-0.150 mcg/kg inclusive. In otherembodiments, the subclinical amount is 0.100 mcg/kg. In otherembodiments, the subclinical amount is 0.150 mcg/kg. In otherembodiments, the subclinical amount is 0.200 mcg/kg. In otherembodiments, the subclinical amount is 0.250 mcg/kg. In otherembodiments, the subclinical amount is 0.300 mcg/kg. In otherembodiments, the subclinical amount is between 0.150-0.400 mcg/kginclusive. In other embodiments, the subclinical amount is between0.150-0.300 mcg/kg inclusive. In other embodiments, the subclinicalamount is between 0.150-0.250 mcg/kg inclusive. In other embodiments,the subclinical amount is between 0.100-0.200 mcg/kg inclusive.

In other embodiments, the subclinical amount of GLP-1 analogue is anamount corresponding to one of the above amounts or ranges for an adult,adjusted per body weight for a pediatric patient. In other embodiments,the GLP-1 analogue is present in a subclinical amount adjusted for apediatric patient, and the insulin is also present in a subclinicalamount adjusted for a pediatric patient, for example an amountcorresponding to 4-12 mg inclusive for an adult patent, adjusted for theweight of the a pediatric patient.

The above amounts may be for exenatide, or in another embodiment, forone of the other GLP-1 analogues known in the art.

In other embodiments, the described dosage form contains exenatide in anamount of between 100-600 mcg inclusive, 100-500 meg inclusive, 100-400meg inclusive, 100-300 meg inclusive, 200-600 mcg inclusive, 200-500 meginclusive, 200-400 meg inclusive, 200-300 meg inclusive, 150-300 mcginclusive, or 150-250 mcg inclusive; together with 8-16 mg insulininclusive. In other embodiments, the described dosage form containsexenatide in an amount of between 100-600 mcg inclusive, 100-500 meginclusive, 100-400 mcg inclusive, 100-300 mcg inclusive, 200-600 mcginclusive, 200-500 mcg inclusive, 200-400 mcg inclusive, 200-300 mcginclusive, 150-300 mcg inclusive, or 150-250 mcg inclusive; togetherwith 8-12 mg insulin inclusive. In other embodiments, the describeddosage form contains exenatide in an amount of between 100-600 mcginclusive, 100-500 mcg inclusive, 100-400 mcg inclusive, 100-300 mcginclusive, 200-600 mcg inclusive, 200-500 mcg inclusive, 200-400 mcginclusive, 200-300 mcg inclusive, 150-300 mcg inclusive, or 150-250 mcginclusive; together with 12-16 mg insulin inclusive. In otherembodiments, the described dosage form contains exenatide in an amountof between 100-600 mcg inclusive, 100-500 mcg inclusive, 100-400 mcginclusive, 100-300 mcg inclusive, 200-600 mcg inclusive, 200-500 mcginclusive, 200-400 mcg inclusive, 200-300 mcg inclusive, 150-300 mcginclusive, or 150-250 mcg inclusive; together with 16-24 mg insulininclusive. In other embodiments, the described dosage form containsexenatide in an amount of between 100-600 mcg inclusive, 100-500 mcginclusive, 100-400 mcg inclusive, 100-300 mcg inclusive, 200-600 mcginclusive, 200-500 meg inclusive, 200-400 mcg inclusive, 200-300 mcginclusive, 150-300 mcg inclusive, or 150-250 mcg inclusive; togetherwith 24-32 mg insulin inclusive. In other embodiments, the describeddosage form contains exenatide in an amount of between 100-600 mcginclusive, 100-500 mcg inclusive, 100-400 mcg inclusive, 100-300 mcginclusive, 200-600 mcg inclusive, 200-500 mcg inclusive, 200-400 mcginclusive, 200-300 mcg inclusive, 150-300 mcg inclusive, or 150-250 mcginclusive; together with 12-16 mg insulin inclusive. In otherembodiments, the described dosage form contains exenatide in an amountof between 100-600 mcg inclusive, 100-500 mcg inclusive, 100-400 mcginclusive, 100-300 mcg inclusive, 200-600 mcg inclusive, 200-500 mcginclusive, 200-400 mcg inclusive, 200-300 mcg inclusive, 150-300 mcginclusive, or 150-250 mcg inclusive; together with 8 mg insulininclusive. In other embodiments, the described dosage form containsexenatide in an amount of between 100-600 mcg inclusive, 100-500 mcginclusive, 100-400 mcg inclusive, 100-300 mcg inclusive, 200-600 mcginclusive, 200-500 mcg inclusive, 200-400 mcg inclusive, 200-300 mcginclusive, 150-300 mcg inclusive, or 150-250 mcg inclusive; togetherwith 12 mg insulin inclusive. In other embodiments, the described dosageform contains exenatide in an amount of between 100-600 mcg inclusive,100-500 mcg inclusive, 100-400 mcg inclusive, 100-300 mcg inclusive,200-600 mcg inclusive, 200-500 mcg inclusive, 200-400 mcg inclusive,200-300 mcg inclusive, 150-300 mcg inclusive, or 150-250 mcg inclusive;together with 16 mg insulin inclusive. In other embodiments, thedescribed dosage form contains exenatide in an amount of between 100-600mcg inclusive, 100-500 mcg inclusive, 100-400 mcg inclusive, 100-300 meginclusive, 200-600 mcg inclusive, 200-500 mcg inclusive, 200-400 mcginclusive, 200-300 mcg inclusive, 150-300 mcg inclusive, or 150-250 mcginclusive; together with 20 mg insulin inclusive. In other embodiments,the described dosage form contains exenatide in an amount of between100-600 mcg inclusive, 100-500 mcg inclusive, 100-400 mcg inclusive,100-300 mcg inclusive, 200-600 mcg inclusive, 200-500 mcg inclusive,200-400 mcg inclusive, 200-300 mcg inclusive, 150-300 mcg inclusive, or150-250 mcg inclusive; together with 24 mg insulin inclusive. In otherembodiments, the described dosage form contains exenatide in an amountof between 100-600 mcg inclusive, 100-500 mcg inclusive, 100-400 mcginclusive, 100-300 mcg inclusive, 200-600 mcg inclusive, 200-500 mcginclusive, 200-400 mcg inclusive, 200-300 mcg inclusive, 150-300 mcginclusive, or 150-250 mcg inclusive; together with 28 mg insulininclusive. In other embodiments, the described dosage form containsexenatide in an amount of between 100-600 mcg inclusive, 100-500 mcginclusive, 100-400 mcg inclusive, 100-300 mcg inclusive, 200-600 mcginclusive, 200-500 mcg inclusive, 200-400 mcg inclusive, 200-300 mcginclusive, 150-300 mcg inclusive, or 150-250 mcg inclusive; togetherwith 32 mg insulin inclusive.

In other embodiments, the described dosage form contains insulin in anamount of 8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20mg inclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 150-300 mcg exenatide inclusive. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20 mginclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 300-450 mcg exenatide inclusive. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20 mginclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 450-600 mcg exenatide inclusive. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20 mginclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 100-150 mcg exenatide inclusive. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20 mginclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 150-200 mcg exenatide inclusive. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20 mginclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 200-250 mcg exenatide inclusive. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20 mginclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 250-300 mcg exenatide inclusive.

In other embodiments, the described dosage form contains insulin in anamount of 8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20mg inclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 100 mcg exenatide inclusive. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20 mginclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 200 mcg exenatide inclusive. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20 mginclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 250 mcg exenatide inclusive. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20 mginclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 300 mcg exenatide inclusive. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20 mginclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 400 mcg exenatide inclusive. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20 mginclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 500 mcg exenatide inclusive. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg inclusive, 8-28 mg inclusive, 8-24 mg inclusive, 8-20 mginclusive, 8-16 mg inclusive, 8-12 mg inclusive, 12-32 mg inclusive,16-32 mg inclusive, 20-32 mg inclusive, 24-32 mg inclusive, 12-24 mginclusive, 16-24 mg inclusive, 12-20 mg inclusive, or 16-20 mginclusive; together with 600 mcg exenatide inclusive.

In other embodiments, the described dosage form contains 8-16 mg insulininclusive and 150-300 mcg. exenatide. In other embodiments, thedescribed dosage form contains 8-12 mg insulin inclusive and 150-300mcg. exenatide. In other embodiments, the described dosage form contains12-16 mg insulin inclusive and 150-300 mcg. exenatide. In otherembodiments, the described dosage form contains 6-16 mg insulininclusive and 150-300 mcg. exenatide.

In other embodiments, the described dosage form contains 8-16 mg insulininclusive and 100-400 mcg. exenatide. In other embodiments, thedescribed dosage form contains 8-12 mg insulin inclusive and 100-400mcg. exenatide. In other embodiments, the described dosage form contains12-16 mg insulin inclusive and 100-400 mcg. exenatide. In otherembodiments, the described dosage form contains 6-16 mg insulininclusive and 100-400 mcg. exenatide.

In other embodiments, the described dosage form contains 8-16 mg insulininclusive and 100-200 mcg. exenatide. In other embodiments, thedescribed dosage form contains 8-12 mg insulin inclusive and 100-200mcg. exenatide. In other embodiments, the described dosage form contains12-16 mg insulin inclusive and 100-200 mcg. exenatide. In otherembodiments, the described dosage form contains 6-16 mg insulininclusive and 100-200 mcg. exenatide.

In other embodiments, the described dosage form contains 8-16 mg insulininclusive and 200-400 mcg. exenatide. In other embodiments, thedescribed dosage form contains 8-12 mg insulin inclusive and 200-400mcg. exenatide. In other embodiments, the described dosage form contains12-16 mg insulin inclusive and 200-400 mcg. exenatide. In otherembodiments, the described dosage form contains 6-16 mg insulininclusive and 200-400 mcg. exenatide.

In other embodiments, the described dosage form contains 8-16 mg insulininclusive and 150-300 mcg. exenatide.

In some embodiments, the patient receiving the described pharmaceuticalcomposition is receiving a small-molecule DM therapeutic agent such asMetformin and/or a thiazolidinedione (TZD). In other embodiments, thepatient is not receiving a small-molecule DM therapeutic agent. Thedescribed compositions are believed to be effective in either instance.

Protease Inhibitors

As used herein, the term “trypsin inhibitor” refers to any agent capableof inhibiting the action of trypsin on a substrate. The ability of anagent to inhibit trypsin can be measured using assays well known in theart. For example, in a typical assay, one unit corresponds to the amountof inhibitor that reduces the trypsin activity by one benzoyl-L-arginineethyl ester unit (BAEE-U). One BAEE-U is the amount of enzyme thatincreases the absorbance at 253 nm by 0.001 per minute at pH 7.6 and 25°C. See, for example, K. Ozawa, M. Laskowski, 1966, J. Biol. Chem.241:3955; and Y. Birk, 1976, Meth. Enzymol. 45:700.

Some trypsin inhibitors known in the art are specific to trypsin, whileothers inhibit trypsin and other proteases such as chymotrypsin. Trypsininhibitors can be derived from animal or vegetable sources: for example,soybean, corn, lima and other beans, squash, sunflower, bovine and otheranimal pancreas and lung, chicken and turkey egg white, soy-based infantformula, and mammalian blood. Trypsin inhibitors can also be ofmicrobial origin: for example, antipain; see, for example, H. Umezawa,1976, Meth. Enzymol. 45, 678. A trypsin inhibitor can also be anarginine or lysine mimic or other synthetic compound: for examplearylguanidine, benzamidine, 3,4-dichloroisocoumarin,diisopropylfluorophosphate, gabexate mesylate, or phenylmethanesulfonylfluoride. As used herein, an arginine or lysine mimic is a compound thatis capable of binding to the P¹ pocket of trypsin and/or interferingwith trypsin active site function.

In certain embodiments, the trypsin inhibitor utilized in methods andcompositions of the present invention is selected from the groupconsisting of lima bean trypsin inhibitor, aprotinin, (a.k.a. pancreatictrypsin inhibitor or basic pancreatic trypsin inhibitor [BPTI]; UniprotNo. P00974 [database accessed on Jan. 2, 2013]), Kazal inhibitor(pancreatic secretory trypsin inhibitor), ovomucoid, Alpha1-antitrypsin, Cortisol binding globulin, Centerin ([SERPINA9/GCET1(germinal centre B-cell-expressed transcript 1)], PI-6 (Sun et al 1995),PI-8 (Sprecher et al 1995), Bomapin, a clade A serpin [for exampleSerpina3 (NCBI Gene ID: 12; database accessed on Dec. 27, 2012),Serpina6 (NCBI Gene ID: 866; database accessed on Dec. 27, 2012),Serpina12 (NCBI Gene ID: 145264; database accessed on Dec. 27, 2012);Serpina10 (NCBI Gene ID: 51156; database accessed on Dec. 27, 2012);Serpina7 (NCBI Gene ID: 6906; database accessed on Dec. 27, 2012);Serpina9 (NCBI Gene ID: 327657; database accessed on Dec. 27, 2012);Serpina11 (NCBI Gene ID: 256394; database accessed on Dec. 27, 2012);Serpina13 (NCBI Gene ID: 388007; database accessed on Dec. 27, 2012);Serpina2 (NCBI Gene ID: 390502; database accessed on Dec. 27, 2012); andSerpina4 (NCBI Gene ID: 5104; database accessed on Dec. 27, 2012)]Yukopin (SerpinB12; Gene ID: 89777; database accessed on Dec. 27, 2012),antipain, benzamidine, 3,4-dichloroisocoumarin,diisopropylfluorophosphate, and gabexate mesylate. In other embodiments,more than one, for example 2, 3, or 4, of the above inhibitors isselected.

A representative precursor sequence of aprotinin is:

(SEQ ID NO: 1) MKMSRLCLSV ALLVLLGTLA ASTPGCDTSN QAKAQRPDFC LEPPYTGPCK ARIIRYFYNA KAGLCQTFVY GGCRAKRNNF KSAEDCMRTC GGAIGPWENL.

Of these 100 residues, residues 1-21 are the signal peptide, 22-35 and94-100 are propeptides, and the mature chain BBI chain is composed ofresidues 36-93 (58 AA).

In other embodiments, the trypsin inhibitor is derived from soybean.Trypsin inhibitors derived from soybean (Glycine max) are readilyavailable and are considered to be safe for human consumption. Theyinclude, but are not limited to, SBTI, KTI (Kunitz Trypsin Inhibitor),for example KTI3, and BBI (Bowman-Birk inhibitor; Uniprot number P01055[database accessed on Jan. 3, 2013]). SBTI is composed of KTI, whichinhibits trypsin, and BBI, which inhibits trypsin and chymotrypsin. Suchtrypsin inhibitors are available for example from Sigma-Aldrich, St.Louis, Mo., USA.

A representative precursor sequence of BBI is:

(SEQ ID NO: 2) MVVLKVCLVL LFLVGGTTSA NLRLSKLGLL MKSDHQHSNDDESSKPCCDQ CACTKSNPPQ CRCSDMRLNS CHSACKSCICALSYPAQCFC VDITDFCYEP CKPSEDDKEN.

Of these 110 residues, residues 1-19 are the signal peptide, 20-39 are apropeptide, and the mature chain BBI chain is composed of residues40-110 (71 AA).

KTI3 has Uniprot number P01070 (database accessed on Jan. 3, 2013). Arepresentative precursor sequence of KTI3 is:

SEQ ID NO: 3) MKSTIFFLFL FCAFTTSYLP SAIADFVLDN EGNPLENGGTYYILSDITAF GGIRAAPTGN ERCPLTVVQS RNELDKGIGTIISSPYRIRF IAEGHPLSLK FDSFAVIMLC VGIPTEWSVVEDLPEGPAVK IGENKDAMDG WFRLERVSDD EFNNYKLVFCPQQAEDDKCG DIGISIDHDD GTRRLVVSKN KPLVVQFQKL DKESLAKKNH GLSRSE 

Of the above sequence, residues 1-24 are the signal peptide, 206-216 area propeptide, and the mature KTI chain is composed of residues 25-205(181 AA).

In other embodiments, a method or oral pharmaceutical compositiondescribed herein utilizes two trypsin inhibitors. In other embodiments,more than two trypsin inhibitors are present. In other embodiments,three trypsin inhibitors are present. In other embodiments, four trypsininhibitors are present. In other embodiments, two of the trypsininhibitors are SBTI and aprotinin. In yet other embodiments, the onlytwo trypsin inhibitors are SBTI and aprotinin. In still otherembodiments, the only two trypsin inhibitors are isolated BBI andisolated aprotinin.

In other embodiments, a chymotrypsin inhibitor is present together witha trypsin inhibitor. In other embodiments, when the chymotrypsininhibitor is also a trypsin inhibitor, another trypsin inhibitor is alsopresent. Non-limiting examples of a trypsin inhibitor and atrypsin/chymotrypsin inhibitor are isolated KTI, which inhibits trypsin,and isolated BBI (Bowman-Birk inhibitor), which inhibits trypsin andchymotrypsin; these are present together in the composition, in someembodiments.

Chelators of Divalent Cations

The chelator of divalent cations utilized in the described methods andcompositions is, in one embodiment, any physiologically acceptablecompound having a high affinity for at least one of calcium, magnesium,and manganese ions. In another embodiment, the chelator is selected fromthe group consisting of citrate or a salt thereof; ethylenediaminetetracetic acid (EDTA) or a salt thereof (for example disodium EDTA andcalcium disodium EDTA); EGTA (ethylene glycol tetraacetic acid) or asalt thereof; diethylene triamine pentaacetic acid (DTPA) or a saltthereof; and BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraaceticacid) or a salt thereof. In other embodiments, one of the above-listedchelators is utilized. In more specific embodiments, the chelator isEDTA.

Oils

Pharmaceutical compositions and methods described herein utilize one ormore oils as the basis of their liquid phase. In certain embodiments,the oil may be any physiologically acceptable oil that is liquid atambient temperature.

In more specific embodiments, the oil comprises an omega-3 fatty acid.In other embodiments, the omega-3 fatty acid is an omega-3polyunsaturated fatty acid. In another embodiment, the omega-3 fattyacid is DHA, an omega-3, polyunsaturated, 22-carbon fatty acid alsoreferred to as 4, 7, 10, 13, 16, 19-docosahexaenoic acid. In anotherembodiment, the omega-3 fatty acid is—linolenic acid (9, 12,15-octadecatrienoic acid). In another embodiment, the omega-3 fatty acidis stearidonic acid (6, 9, 12, 15-octadecatetraenoic acid). In anotherembodiment, the omega-3 fatty acid is eicosatrienoic acid (ETA; 11, 14,17-eicosatrienoic acid). In another embodiment, the omega-3 fatty acidis eicsoatetraenoic acid (8, 11, 14, 17-eicosatetraenoic acid). In oneembodiment, the omega-3 fatty acid is eicosapentaenoic acid (EPA; 5, 8,11, 14, 17-eicosapentaenoic acid). In another embodiment, the omega-3fatty acid is eicosahexaenoic acid (also referred to as 5, 7, 9, 11, 14,17-eicosahexaenoic acid). In another embodiment, the omega-3 fatty acidis docosapentaenoic acid (DPA; 7, 10, 13, 16, 19-docosapenatenoic acid).In another embodiment, the omega-3 fatty acid is tetracosahexaenoic acid(6, 9, 12, 15, 18, 21-tetracosahexaenoic acid).

In other embodiments, the oil is a naturally-occurring oil comprising anomega-3 fatty acid. In more specific embodiments, the oil is selectedfrom the group consisting of a fish oil, canola oil, flaxseed oil, algaloil and hemp seed oil. In more specific embodiments, the oil is a fishoil.

Several types of fish oil have been tested in the compositions describedherein and have all been found to work equally well.

Representative Specific Formulations

In still more specific embodiments, a liquid formulation utilized in thedescribed method or composition comprises insulin, exenatide, Gelucire44/14, EDTA, SBTI, aprotinin, and fish oil. In other embodiments, theliquid formulation consists essentially of insulin, exenatide, Gelucire44/14, EDTA, SBTI, aprotinin, and fish oil. “Consists essentially of”for these purposes indicates that the liquid formulation does notcontain any other components that appreciably affect its physiologicalcharacteristics. In other embodiments, the liquid formulation consistsof insulin, exenatide, Gelucire 44/14, EDTA, SBTI, aprotinin, and fishoil. In other, even more specific embodiments, the amounts of insulin,exenatide, EDTA, SBTI, aprotinin, and fish oil per dosage form are 8-16mg, 150-300 mcg, 100-200 mg, 50-100 mg, 20-30 mg, and 0.4-0.7 ml,respectively, and the amount of Gelucire 44/14 is 8-16%. In still morespecific embodiments, the amounts of insulin, exenatide, EDTA, SBTI,aprotinin, and fish oil per dosage form are 8-16 mg, 150-300 mcg, 150mg, 75 mg, 24 mg, and 0.5-0.7 ml, respectively, and the amount ofGelucire 44/14 is 8-16%. In other embodiments, the above compositionfurther comprises a non-ionic detergent. In more specific embodiments,the non-ionic detergent is a polysorbate-based detergent. In even morespecific embodiments, the polysorbate-based detergent is polysorbate 80.Preferably, the polysorbate 80 constitutes 3-10% weight/weight inclusiveof the oil-based liquid formulation. In other embodiments, the abovecomposition is coated by a coating that resists degradation in thestomach.

In still more specific embodiments, a liquid formulation utilized in thedescribed method or composition comprises insulin, exenatide, aself-emulsifying component, EDTA, SBTI, aprotinin, and fish oil. Inother embodiments, the liquid formulation consists essentially ofinsulin, exenatide, a self-emulsifying component, EDTA, SBTI, aprotinin,and fish oil. “Consists essentially of” for these purposes indicatesthat the liquid formulation does not contain any other components thatappreciably affect its physiological characteristics. In otherembodiments, the liquid formulation consists of insulin, exenatide, aself-emulsifying component, EDTA, SBTI, aprotinin, and fish oil. Inother, even more specific embodiments, the amounts of insulin,exenatide, EDTA, SBTI, aprotinin, and fish oil per dosage form are 8-16mg, 150-300 mcg, 100-200 mg, 50-100 mg, 20-30 mg, and 0.4-0.7 ml,respectively, and the amount of self-emulsifying component is 8-16%. Instill more specific embodiments, the amounts of insulin, exenatide,EDTA, SBTI, aprotinin, and fish oil per dosage form are 8-16 mg, 150-300mcg, 150 mg, 75 mg, 24 mg, and 0.5-0.7 ml, respectively, and the amountof self-emulsifying component is 8-16%. In other embodiments, the abovecomposition further comprises a non-ionic detergent. In more specificembodiments, the non-ionic detergent is a polysorbate-based detergent.In even more specific embodiments, the polysorbate-based detergent ispolysorbate 80. Preferably, the polysorbate 80 constitutes 3-10%weight/weight inclusive of the oil-based liquid formulation. In otherembodiments, the above composition is coated by a coating that resistsdegradation in the stomach.

“Weight/weight” percentages referred to herein utilize the amount of oilbase in the formulation, for example fish oil, as the denominator; thus,60 mg of Gelucire in 500 mg fish oil is considered as 12% w/w,regardless of the weight of the other components. Similarly, 50 mg.Tween-80 mixed with 500 mg fish oil is considered as 10% Tween-80.

In other embodiments, a liquid formulation utilized in the describedmethod or composition is water-free. If more than one liquid formulationis present, for example in a multi-component composition, each liquidformulation may be water-free. “Water-free” refers, in certainembodiments, to a formulation into which no aqueous components have beenintentionally added. It does not preclude the presence of trace amountsof water that have been absorbed from the atmosphere into the componentsthereof. In another embodiment, the liquid formulation is free ofaqueous components. If more than one liquid formulation is present, forexample in a multi-component composition, each liquid formulation may befree of aqueous components. In yet other embodiments, one or more oilsare the only liquid components of each of the one or more liquidformulations. In yet another embodiment, fish oil is the only liquidcomponent of each of the one or more liquid formulations.

Coatings

Those of skill in the art will appreciate, given the present disclosure,that various pH-sensitive coatings may be utilized in the describedmethods and compositions. In certain embodiments, any coating thatinhibits digestion of the composition in the stomach of a subject may beutilized. Typically, such coatings will not dissolve in human gastricjuices within 2 hours, and will dissolve within 30 minutes in duodenalfluid.

In other embodiments, the coating comprises a biodegradablepolysaccharide. In other embodiments, a hydrogel is utilized. In otherembodiments, the coating comprises one of the following excipients:chitosan, an aquacoat ECD coating, an azo-crosslinked polymer, celluloseacetate phthalate, cellulose acetate trimellitate (CAT), celluloseacetate butyrate, hydroxypropylmethyl cellulose phthalate, or poly vinylacetate phthalate.

In other embodiments, a timed-release system such as Pulsincap™ isutilized.

In preferred embodiments, the coated dosage forms described hereinrelease the core (containing the oil-based formulation) when pH reachesthe range found in the intestines, which is alkaline relative to that inthe stomach. In more specific embodiments, the coating comprises apH-sensitive polymer. In various embodiments, either mono-layer ormulti-layer coatings may be utilized.

In one embodiment, the coating is an enteric coating. Methods forenteric coating are well known in the art (see, for example, Siepmann Fet al 2005). In more specific embodiments, a Eudragit™ coating isutilized as the enteric coating. Eudragit® coatings are acrylicpolymers, the use of which is well known in the art.

In another embodiment, microencapsulation is used as a stomach-resistantcoating in the compositions described herein. Methods formicroencapsulation are well known in the art, and are described interalia in United States Patent Application Publication No. 2011/0305768,which is incorporated by reference herein.

In other embodiments, the coating is a capsule. Gelatin capsules aremost preferred. Methods for inserting an oil-based formulation into agelatin capsule are well known in the art.

Pharmaceutical Compositions and Methods of Making Same

In another aspect a pharmaceutical composition described herein isprovided for treating diabetes mellitus, for example Type 2 diabetesmellitus, in a human.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for treatingdiabetes mellitus in a human.

Still another aspect provides a method for treating diabetes mellitus ina human, the method comprising the optional step of selecting a subjectby diagnosing diabetes mellitus, followed by the step of administeringto a subject in need of such treatment a pharmaceutical compositiondescribed herein, thereby treating diabetes mellitus in a human.

Still another aspect provides a method of manufacturing a pharmaceuticalcomposition, comprising the steps of melting a waxy, self-emulsifyingcomponent; adding the molten component to fish oil, optionally coolingthe resulting mixture; adding to the oil EDTA, SBTI in powder form,aprotinin in powder form, crystalline insulin, and exenatide in powderform, in some embodiments in the order listed; and mixing andhomogenizing the resulting liquid, in some embodiments on a roller mill.

Yet another aspect provides a method of manufacturing a pharmaceuticalcomposition, comprising the steps of melting a waxy, self-emulsifyingcomponent; adding the molten component to fish oil, optionally coolingthe resulting mixture; adding to the oil EDTA, isolated BBI in powderform, isolated KTI3 in powder form, crystalline insulin, and exenatidein powder form, in some embodiments in the order listed; and mixing andhomogenizing the resulting liquid, in some embodiments on a roller mill.

Yet another aspect provides a method of manufacturing a pharmaceuticalcomposition, comprising the steps of melting a waxy, self-emulsifyingcomponent; adding the molten component to fish oil, optionally coolingthe resulting mixture; adding to the oil EDTA, isolated KTI3 in powderform, aprotinin in powder form, crystalline insulin, and exenatide inpowder form, in some embodiments in the order listed; and mixing andhomogenizing the resulting liquid, in some embodiments on a roller mill.

Yet another aspect provides a use of a combination of ingredientsdescribed hereinabove in the preparation of a medicament for treatingunstable diabetes, also known as glycemic lability (Ryan et al, 2004) ina human. Yet another aspect provides a method for treating unstablediabetes, the method comprising the step of administering to a subjectin need of such treatment a pharmaceutical composition describedhereinabove, thereby treating unstable diabetes.

Provided in another embodiment is a use of a combination of ingredientsdescribed hereinabove in the preparation of a medicament for treating anelevated fasting blood glucose level in a human. Provided in anotherembodiment is a method for treating an elevated fasting blood glucoselevel, the method comprising the step of administering to a subject inneed of such treatment a pharmaceutical composition describedhereinabove, thereby treating an elevated fasting blood glucose. Incertain preferred embodiments, the subject is a human subject. Invarious embodiments, elevated fasting blood glucose is considered toexist in a subject having a glycated hemoglobin [HgAlc] level of 8-10%,or a fasting plasma sugar level from 100 to 125 mg/dL, or 5.6 to 6.9mmol/L.

Provided herein, in another embodiment, is a method of treating elevatedtotal cholesterol, the method comprising the step of administering to asubject in need of such treatment a pharmaceutical composition describedhereinabove, thereby treating elevated total cholesterol.

Another aspect provides a method of treating hypertriglyceridemia, themethod comprising the step of administering to a subject in need of suchtreatment a pharmaceutical composition described hereinabove, therebytreating hypertriglyceridemia.

Another aspect provides a method of treating elevated serumapolipoprotein B (ApoB), the method comprising the step of administeringto a subject in need of such treatment a pharmaceutical compositiondescribed hereinabove.

Another aspect provides a method of treating elevated totalcholesterol/HDL ratio, the method comprising the step of administeringto a subject in need of such treatment a pharmaceutical compositiondescribed hereinabove.

Another aspect provides a method of treating an elevated apolipoproteinB/apolipoprotein A1 ratio, the method comprising the step ofadministering to a subject in need of such treatment a pharmaceuticalcomposition described hereinabove, thereby treating an elevatedapolipoprotein B/apolipoprotein A1 ratio.

Methods for measuring each of the aforementioned lipid parameters arewell known to those skilled in the art. Exemplary methods are describedinter alia in Chiquette E et al and Martinez-Colubi M et al.

Another aspect provides a method of treating an impaired insulin-inducedenhancement of vasodilator responses in a subject with metabolicsyndrome, the method comprising the step of administering to a subjectin need of such treatment a pharmaceutical composition describedhereinabove, thereby treating an impaired insulin-induced enhancement ofvasodilator responses in a subject with metabolic syndrome. Methods formeasuring insulin-induced vasodilator responses are known in the art,and include, for example, measuring blood flow responses (for example inthe forearm) to acetylcholine (ACh) and sodium nitroprusside (SNP)(Tesauro et al).

Metabolic syndrome is considered to be present if at least three of thefollowing factors are present:

-   -   1. A large waistline (abdominal obesity).    -   2. A high triglyceride level (or a high triglyceride level in        the absence of medicine to treat high triglycerides).    -   3. A low HDL cholesterol level (or low HDL cholesterol in the        absence of medicine to treat low HDL cholesterol).    -   4. Hypertension (or hypertension in the absence of medicine to        treat hypertension.    -   5. High fasting blood sugar (or high fasting blood sugar in the        absence of medicine to treat high fasting blood sugar).

Another aspect provides a method of treating non-alcoholicsteatohepatitis in a subject with metabolic syndrome, the methodcomprising the step of administering to a subject in need of suchtreatment a pharmaceutical composition described herein, therebytreating non-alcoholic steatohepatitis in a subject with metabolicsyndrome. Methods of diagnosing and measuring non-alcoholicstcatohepatitis are well known in the art, and include, for example,measuring plasma aspartate transaminase (AST) levels, plasma alaninetransaminase (ALT) levels, hepatic mRNA levels of genes involved inlipogenesis, and diacylglycerol acyltransferase-2 (DGAT2) levels in theliver (Miyashita T et al).

Another aspect provides a method of treating elevated total cholesterol,the method comprising the step of administering to a subject in need ofsuch treatment an oral, oil-based liquid formulation, the oil-basedliquid formulation comprising an insulin, one or, in another embodimentmore than one, trypsin inhibitor, and a chelator of divalent cations,wherein the oil-based liquid formulation is surrounded by a coating orcapsule that resists degradation in the stomach. Another aspect providesa method of treating elevated total cholesterol, the method comprisingthe step of administering to a subject in need of such treatment anoral, oil-based liquid formulation, the oil-based liquid formulationcomprising a GLP-1 analogue, one or, in another embodiment more thanone, trypsin inhibitor, and a chelator of divalent cations, wherein theoil-based liquid formulation is surrounded by a coating or capsule thatresists degradation in the stomach. The oil, insulin, GLP-1 analogue,trypsin inhibitor(s), chelator, coating, and other optional ingredientsof the above compositions may be any described herein; each alternativemay be combined freely to form discrete embodiments of the inventiondisclosed herein.

Another aspect provides a method of treating hypertriglyceridemia, themethod comprising the step of administering to a subject in need of suchtreatment an oral, oil-based liquid formulation, the oil-based liquidformulation comprising an insulin, one or, in another embodiment morethan one, trypsin inhibitor, and a chelator of divalent cations, whereinthe oil-based liquid formulation is surrounded by a coating or capsulethat resists degradation in the stomach. Another aspect provides amethod of treating hypertriglyceridemia, the method comprising the stepof administering to a subject in need of such treatment an oral,oil-based liquid formulation, the oil-based liquid formulationcomprising a GLP-1 analogue, one or, in another embodiment more thanone, trypsin inhibitor, and a chelator of divalent cations, wherein theoil-based liquid formulation is surrounded by a coating or capsule thatresists degradation in the stomach. The oil, insulin, GLP-1 analogue,trypsin inhibitor(s), chelator, coating, and other optional ingredientsof the above compositions may be any described herein; each alternativemay be combined freely to form discrete embodiments of the inventiondisclosed herein.

Another aspect provides a method of treating elevated serum ApoB, themethod comprising the step of administering to a subject in need of suchtreatment an oral, oil-based liquid formulation, the oil-based liquidformulation comprising an insulin, one or, in another embodiment morethan one, trypsin inhibitor, and a chelator of divalent cations, whereinthe oil-based liquid formulation is surrounded by a coating or capsulethat resists degradation in the stomach. Another aspect provides amethod of treating elevated serum ApoB, the method comprising the stepof administering to a subject in need of such treatment an oral,oil-based liquid formulation, the oil-based liquid formulationcomprising a GLP-1 analogue, one or, in another embodiment more thanone, trypsin inhibitor, and a chelator of divalent cations, wherein theoil-based liquid formulation is surrounded by a coating or capsule thatresists degradation in the stomach. The oil, insulin, GLP-1 analogue,trypsin inhibitor(s), chelator, coating, and other optional ingredientsof the above compositions may be any described herein; each alternativemay be combined freely to form discrete embodiments of the inventiondisclosed herein.

Another aspect provides a method of treating an elevated totalcholesterol/HDL ratio, the method comprising the step of administeringto a subject in need of such treatment an oral, oil-based liquidformulation, the oil-based liquid formulation comprising an insulin, oneor, in another embodiment more than one, trypsin inhibitor, and achelator of divalent cations, wherein the oil-based liquid formulationis surrounded by a coating or capsule that resists degradation in thestomach. Another aspect provides a method of treating an elevated totalcholesterol/HDL ratio, the method comprising the step of administeringto a subject in need of such treatment an oral, oil-based liquidformulation, the oil-based liquid formulation comprising a GLP-1analogue, one or, in another embodiment more than one, trypsininhibitor, and a chelator of divalent cations, wherein the oil-basedliquid formulation is surrounded by a coating or capsule that resistsdegradation in the stomach. The oil, insulin, GLP-1 analogue, trypsininhibitor(s), chelator, coating, and other optional ingredients of theabove compositions may be any described herein; each alternative may becombined freely to form discrete embodiments of the invention disclosedherein.

Another aspect provides a method of treating an elevated apolipoproteinB/apolipoprotein A1 ratio, the method comprising the step ofadministering to a subject in need of such treatment an oral, oil-basedliquid formulation, the oil-based liquid formulation comprising aninsulin, one or, in another embodiment more than one, trypsin inhibitor,and a chelator of divalent cations, wherein the oil-based liquidformulation is surrounded by a coating or capsule that resistsdegradation in the stomach. Another aspect provides a method of treatingan elevated apolipoprotein B/apolipoprotein A1 ratio, the methodcomprising the step of administering to a subject in need of suchtreatment an oral, oil-based liquid formulation, the oil-based liquidformulation comprising a GLP-1 analogue, one or, in another embodimentmore than one, trypsin inhibitor, and a chelator of divalent cations,wherein the oil-based liquid formulation is surrounded by a coating orcapsule that resists degradation in the stomach. The oil, insulin, GLP-1analogue, trypsin inhibitor(s), chelator, coating, and other optionalingredients of the above compositions may be any described herein; eachalternative may be combined freely to form discrete embodiments of theinvention disclosed herein.

Another aspect provides a method of treating an impaired insulin-inducedenhancement of vasodilator responses in a subject with metabolicsyndrome, the method comprising the step of administering to a subjectin need of such treatment an oral, oil-based liquid formulation, theoil-based liquid formulation comprising an insulin, one or, in anotherembodiment more than one, trypsin inhibitor, and a chelator of divalentcations, wherein the oil-based liquid formulation is surrounded by acoating or capsule that resists degradation in the stomach.

Another aspect provides a method of treating an impaired insulin-inducedenhancement of vasodilator responses in a subject with metabolicsyndrome, the method comprising the step of administering to a subjectin need of such treatment an oral, oil-based liquid formulation, theoil-based liquid formulation comprising a GLP-1 analogue, one or, inanother embodiment more than one, trypsin inhibitor, and a chelator ofdivalent cations, wherein the oil-based liquid formulation is surroundedby a coating or capsule that resists degradation in the stomach. Theoil, insulin, GLP-1 analogue, trypsin inhibitor(s), chelator, coating,and other optional ingredients of the above compositions may be anydescribed herein; each alternative may be combined freely to formdiscrete embodiments of the invention disclosed herein.

Another aspect provides a method of treating non-alcoholicsteatohepatitis in a subject with metabolic syndrome, the methodcomprising the step of administering to a subject in need of suchtreatment an oral, oil-based liquid formulation, the oil-based liquidformulation comprising an insulin, one or, in another embodiment morethan one, trypsin inhibitor, and a chelator of divalent cations, whereinthe oil-based liquid formulation is surrounded by a coating or capsulethat resists degradation in the stomach. Another aspect provides amethod of treating non-alcoholic steatohepatitis in a subject withmetabolic syndrome, the method comprising the step of administering to asubject in need of such treatment an oral, oil-based liquid formulation,the oil-based liquid formulation comprising a GLP-1 analogue, one or, inanother embodiment more than one, trypsin inhibitor, and a chelator ofdivalent cations, wherein the oil-based liquid formulation is surroundedby a coating or capsule that resists degradation in the stomach. Theoil, insulin, GLP-1 analogue, trypsin inhibitor(s), chelator, coating,and other optional ingredients of the above compositions may be anydescribed herein; each alternative may be combined freely to formdiscrete embodiments of the invention disclosed herein.

Wherever alternatives for single separable features such as, forexample, a insulin protein or dosage thereof, a GLP-1 analogue of dosagethereof, a protease inhibitor, a chelator, an emulsifier, or a coatingare laid out herein as “embodiments”, it is to be understood that suchalternatives may be combined freely to form discrete embodiments of theinvention disclosed herein.

With respect to the jurisdictions allowing it, all patents, patentapplications, and publications mentioned herein, both supra and infra,are incorporated herein by reference.

The invention is further illustrated by the following examples and thefigures, from which further embodiments and advantages can be drawn.These examples are meant to illustrate the invention but not to limitits scope.

EXPERIMENTAL DETAILS SECTION Example 1: Identification of EffectiveEmulsifiers for Homogenous Therapeutic Protein/Fish Oil Preparations

Previous formulations of insulin in fish oil were found to exhibitprecipitation with the passage of time; they were thus inconvenient forlarge-scale pharmaceutical dosage form preparation. New formulationscontaining 3.375 g. SBTI per 22.5 g. of fish oil and containing thefollowing emulsifiers were tested: lecithin (trial sequence 1),Polysorbate 80 (Tween-80) (sequence 2), or Gelucire® 44/14 (sequence 3),alone or in combination with each other or glycerol monostearate (GMS)(FIG. 1). Subsequently, the most promising formulations (indicated withan asterisk) were produced again by melting the Gelucire® (which was awaxy solid as ambient temperature), then adding it to the fish oil.After cooling this mixture, the solid components were added in powderform in the following order: EDTA, SBTI, aprotinin, and insulin; and theresulting liquid was mixed and homogenized on a roller mill.

Example 2: In Vivo Testing of Various Emulsifier Formulations

Materials and Experimental Methods

Formulations

The formulations tested in Experiments 2A and 2B are shown below inTable 1. Percentages of emulsifiers are expressed as weight/weight withrespect to the weight of the liquids present.

TABLE 1 Formulation name Emulsifiers Other ingredients Experiment 2A GMS2% GMS 2% only 3.375 g. SBTI, 6.75 mg EDTA, 1.08 mg aprotinin, 0.39 mg.human insulin, 22.5 g. of fish oil 2%-2% 2% GMS, 2% lecithin Same asabove. 2%-10% 2% GMS, 10% lecithin Same as above. 10% lec. 10% lecithinonly Same as above. Experiment 2B A 5% lecithin, 2% GMS Same as above. B3% lecithin, 12% Gelucire 44/14 Same as above. C 6% lecithin, 12%Gelucire 44/14 Same as above. D 5% Tween-80, 12% Gelucire Same as above.44/14 E 10% Tween-80, 12% Gelucire Same as above. 44/14 F 12% Gelucire44/14 only Same as above.

Husbandry

Animal health: Only healthy pigs, as certified by a clinicalveterinarian, were used for the study. Housing: Solitary when with CVCand grouped at other times. Bedding: Concrete+woodchips. Illumination:12-12 h light cycle. Temperature: 19-25° C.

Identification

Each animal was uniquely identified via ear tags.

Experimental Design

Animals were deprived of food 24-36 hours prior to testing. Access towater was ad libitum.

Animals were anesthetized with 20 mg/kg ketamine+2 mg/kg xylazine.Fasting and anesthetized pigs were positioned on their left side beforeliquid formulations were administered under endoscopic guidance,directly to the duodenum. After injection of the formulation, 1 mL fishoil was injected, followed by 10 mL air, to flush the apparatus, therebyensuring administration of the entire formulation. Pigs were thenreturned to their pens to allow for full recovery from the anesthetictreatment, which required 10-15 min. Blood samples (0.5 mL of which wereanalyzed) were periodically drawn from the central line catheter (CVC)over the ensuing 240-min monitoring period. Blood glucose concentrationswere determined from each sample, at each time point. Piglets wereintravenously treated with gentamycin (100 mg/10 kg) after everyexperiment day to avoid infection. In cases where glucose concentrationsdropped below 30 mg/dL, piglets were served commercial pig chow, andglucose concentrations were monitored for an additional 30 minutesthereafter.

A washout period of at least 2 days was enforced between test days.

Results

10 insulin-fish oil formulations with different emulsifiers were testedfor in-vivo activity on blood glucose levels in 2 separate experiments.Results are described below.

TABLE 2 Results of Experiment 2A. The three numbers in each box indicatebaseline value, lowest value, and end (20 mg/dL). “Low” indicates avalue of less than 20 mg/dL. Pig 5 Formulation Pig 1 Pig2 Pig3 Pig4(stoma) Score GMS 2% 78 → 48 → 72 → 1ow → 63 → 23 → 67 → 32 → 55 → 21 →3 65 low 28 60 58 2% GMS, 82 → Low → 78 → 45 → 85 → 30 → 78 → 23 → 2 2%lecithin 65 76 68 61 2% GMS, 76 → Low → 76 → low → 76 → low → 74 → low →4 10% 77 21 low 70 lecithin 10% 51 → 63 → 50 → low → 57 → low → 61 → low→ 5 lecithin 71 low low low

The results of Experiment 2B are indicated in FIG. 2.

Example 3: An Oral Insulin/Oral Exenatide Combination Sharply ReducesBlood Glucose Levels and Prevents Postprandial Glucose Excursions

Materials and Experimental Methods

Animals

Healthy 25-30 kg, 3-4 month-old pigs were used.

Experimental Design

Pigs were deprived of food for 24-36 hours before the start of thestudy. CVCs were replaced every five days, unless circumstances requiredearlier replacement. Animals were anesthetized by isofluraneadministration through a mask before tracheal intubation and respiratorhook-up (2 L O₂ per minute and 3-5% isoflurane, when required), thenwere positioned on their left side and capsules were administereddirectly into the duodenum, using an endoscopic basket under endoscopicguidance. When both oral insulin and oral exenatide were administered,oral exenatide was delivered first, followed by oral insulin within 2-10minutes. Pigs were returned to their pens to allow for full recoveryfrom the anesthetic treatment and 30 minutes after capsuleadministration were given 10 g/kg piglet commercial milk powder(Denkapig Premium (Denkavit) prepared in an equal volume of water. Mealswere typically consumed within 7-15 minutes. Blood samples wereperiodically drawn from the CVC for glucose concentration testing overthe ensuing 240-min monitoring period. Piglets were intravenouslytreated with gentamycin (100 mg/10 kg) after every experiment day toavoid infection. In cases where glucose concentrations dropped below 30mg/dL, piglets were served commercial pig chowder and glucoseconcentrations were monitored for an additional 30 minutes thereafter.Control pigs were untreated. Three pigs were used, each of whom receivedeach formulation a number of times. The number of tests were n=5 fororal insulin, n=5 for oral exenatide, n=7 for the combination, and n=6for the control.

Formulations

The oral insulin formulation contained 8 mg insulin, 12% Gelucire 44/14,150 mg EDTA, 75 mg SBTI, and 24 mg aprotinin in 0.5-0.7 mL fish oil. Theliquid was coated by a soft-gel, enteric-coated capsule. The dosage formwas manufactured by Swiss Caps AG.

The oral exenatide formulation contained 150 microgram (mcg) exenatide,150 mg EDTA, 75 mg SBTI, and 24 mg aprotinin in 0.5-0.7 mL fish oil. Theliquid was coated by a soft-gel, enteric-coated capsule. The dosage formwas manufactured by Swiss Caps AG.

Statistical Analysis

p-values were calculated using a paired, one-tailed t-test.

Results

As expected, placebo-treated pigs experienced glucose excursionsfollowing caloric intake. Pigs treated with oral exenatide 30 minutesbefore caloric intake completely avoided the glucose excursionsthroughout the 150 minutes following caloric intake (FIG. 3). Oralinsulin also curbed glucose excursions, but did not fully prevent a risein blood glucose concentrations throughout the entire monitoring period.A sharply greater effect was observed upon combined treatment with oralexenatide and oral insulin. In addition to fully preventing glucoseexcursions, the combined treatment reduced glucose concentrations to alow of more than 50% below baseline values during the entire period fromthe 70-minute timepoint until the end of the 180-minute monitoringsession. The difference in blood glucose levels was statisticallysignificant compared to no treatment or treatment with insulin orexenatide alone (p-values: combination vs. control: 5.3×10⁻⁵;combination vs. oral insulin: 0.00015, and combination vs. oralexenatide: 1.6×10⁻⁵).

Example 4: Testing of an Oral Insulin/Oral Exenatide Combination inHuman Subjects

Volunteers, either diabetic or non-diabetic, are administered prior to ameal one or more dosage forms having a pH-sensitive coating or capsulecontaining a liquid formulation containing one or more proteaseinhibitors, EDTA, insulin and exenatide. One representative formulationis 50-200 mg. per capsule SBTI; 20-30 mg. per capsule Aprotinin; 75-200mg EDTA; 8-32 mg. per capsule insulin; 150-600 mcg. per capsuleexenatide; and 0.5-0.7 ml fish oil, optionally with an emulsifier (e.g.8-20% Gelucire® 44/14). In other experiments, subjects are administeredtwo encapsulated liquid formulations, one containing proteaseinhibitor(s), EDTA, and insulin, and the other containing proteaseinhibitor(s), EDTA, and exenatide. One representative formulation is afirst capsule containing one or more protease inhibitors, EDTA, andinsulin in a liquid formulation, optionally with an emulsifier (e.g.Gelucire® 44/14), together with a second capsule containing proteaseinhibitors, emulsifier, EDTA, and exenatide in a liquid formulation,optionally with an emulsifier (e.g. Gelucire® 44/14).

Subjects are challenged with a 75 g oral glucose load after capsuleadministration (for example 30-60 minutes later) and relevantphysiological parameters are subsequently monitored by collection ofblood samples.

The following protocol is an exemplary protocol that may be used. Thoseskilled in the art will appreciate that non-essential details of theprotocol may be modified without compromising its ability to provide thedesired information.

Overview

Stage I:

Stage I consists of two segments. Segment 1 will assess the safety,tolerability and the pharmacokinetics/pharmacodynamics (PK/PD) ofescalating doses of oral exenatide in eight (8) healthy volunteers. Inthe first segment, the two lower doses of oral exenatide (150 and 300 μgexenatide) will be randomly tested among all healthy, fasting subjects.If deemed safe, further dose escalation (450 and 600 μg exenatide) willbe authorized in Segment 2. The highest tolerable dose will then beadministered to the subjects 60 minutes before a standard meal (Visit5).

Stage II:

This stage will assess the T2DM patient response to escalating doses ofexenatide when delivered 60 min before a standard meal. Placebo controlswill be included in the study, as will treatment with an active controlof Byetta® (5 μg) subcutaneously delivered 30 min before a standardmeal. In addition, the T2DM subjects will be treated with an oralinsulin capsule containing 16 mg of insulin and with a combination oforal insulin/oral exenatide, at two independent study visits, 60 minutesbefore a standard meal.

In addition to exenatide and/or insulin, all formulations contain 75 mgSBTI, 24 mg aprotinin, and 150 mg EDTA, all in fish oil, inenteric-coated capsules.

Interpretation:

The AUC of glucose reductions and insulin excursion will be calculatedand compared between the different treatments.

Study Population:

Healthy: Male, healthy individuals.

T2DM: The presence of T2DM is determined by WHO criteria. Subjects maybe of either gender, between the ages of 18 and 60, with stable glycemiccontrol, optionally on oral antidiabetes medication (e.g. metforminand/or TZDs), and not pregnant or breastfeeding. Subjects will continuetheir regular medications and dosing regimen up to the night prior tothe study. Patients will resume their regular medication aftercompleting each study session (same day). Other than diabetes, subjectsshould be in general good health, with stable liver enzyme levels (below2× the upper normal range).

Procedures

Stage I:

Segment I of the treatment phase will consist of two visits, evaluating150 μg and 300 μg oral exenatide versus placebo treatment in healthysubjects, administered in the morning after an 8-hr fast. Further doseescalation to Segment II will be authorized if no serious side effectsand safety and tolerability are demonstrated. In Segment II, fastingsubjects will be administered 450 and/or 600 μg oral exenatide and/orplacebo to fasting individuals (Appendix 1). The highest tolerable dosewill then be delivered 60 minutes before intake of a standard meal.

Each dosing will be followed by at least a 72-hour washout period.

Screening Phase:

The following evaluations may be performed:

-   -   Medical history    -   Physical examination    -   Medication history    -   ECG    -   Vital signs (blood pressure, heart rate). Vital signs will be        measured in the sitting position after at least 5 minutes of        rest.    -   Clinical laboratory evaluations (chemistry, hematology, HgA1C)

Treatment Phase:

-   1. Blood samples will be obtained at −45, −30, −15, 0, 15, 30, 45,    60, 75, 90, 105, 120, 135, 150 min, where time=0 is the time at    which the capsule is ingested, and tested for glucose, insulin,    c-peptide, as well as GLP-1 analogue content.-   2. A standard meal will be served 60 minutes after oral exenatide    administration to healthy patients at visit 5 only.-   3. Gastrointestinal symptoms may be assessed.

Stage II:

300 and 600 micrograms exenatide, or the two highest tolerable dosesdetermined in Stage I, will be administered in parallel and incombination with 16 mg insulin, for example as 16 mg combined with 300microgram, 60 minutes before a standard meal. On study visits where aninjection of Byetta® (5 μg) is administered, a meal will be served 30minutes after dosing.

Screening Phase:

Same as for Stage 1.

Treatment Phase:

1. Blood samples will be drawn at −15, 0, 15, 30, 45, 60, 75, 90, 105,120, 135, 150, and 240 min, where time=0 is the time at which thecapsule is ingested, and tested for glucose, insulin, c-peptide, as wellas GLP-1 analogue levels (C_(max), T_(max) and/or AUC)

2. A standard meal will be served 60 minutes after oral dosing or 30minutes after subcutaneous administration of Byetta®.

3. Gastrointestinal symptoms may be assessed.

Treatments (delivered in a randomized order):

-   -   a. 300 or 600 mcg oral exenatide, or placebo, or 5 mcg        subcutaneous exenatide    -   b. 300 or 600 mcg oral exenatide, or placebo, or 5 mcg        subcutaneous exenatide    -   c. 300 or 600 mcg oral exenatide, or placebo, or 5 mcg        subcutaneous exenatide    -   d. 16 mg oral insulin    -   e. 300 mcg oral exenatide+16 mg oral insulin.

Safety Evaluations:

-   -   Adverse events will be collected throughout the study beginning        from the time the subject signs the consent form until the end        of study evaluations    -   Concomitant medications/therapies will be recorded throughout        the duration of study, beginning from the time the subject signs        the informed consent.    -   Vital signs (blood pressure, heart rate) will be measured in the        sitting position after at least 5 minutes of rest at the        following times:        -   Screening        -   Approximately 20 minutes prior to study drug administration        -   Approximately 1 and 2.5 hours post study drug administration    -   Clinical laboratory evaluations (chemistry, hematology) will be        performed at screening and at end of study or early        discontinuation.    -   Physical examinations at screening and end of study or early        discontinuation.    -   Electrocardiograms (ECG) at screening. An ECG will be performed        at end of study or early discontinuation, only if the        investigator deems it necessary.

Example 5: Testing of an Oral Insulin/Oral Exenatide Combination inTreatment of Unstable Diabetes

Subjects with elevated fasting glucose levels (for example, subjectshaving a glycated hemoglobin [HgAlc] level of 8-10%, or a fasting plasmasugar level from 100 to 125 mg/dL or 5.6 to 6.9 mmol/L).) are monitoredfor several days using a blinded continuous glucose monitor (CGM) toestablish a baseline. During several subsequent days, they areadministered a formulation described herein, optionally prior to meals.Blinded CGM is performed to determine the efficacy of the formulations.

In the claims, the word “comprise”, and variations thereof such as“comprises”, “comprising”, and the like indicate that the componentslisted are included, but not generally to the exclusion of othercomponents.

REFERENCES

-   Chiquette E et al. Treatment with exenatide once weekly or twice    daily for 30 weeks is associated with changes in several    cardiovascular risk markers. Vase Health Risk Manag. 2012; 8:621-9.-   Eldor R, Kidron M, Arbit E. A single-bling, two-period study to    assess the safety and pharmacodynamics of an orally delivered GLP-1    analog (exenatide) in healthy subjects.-   American Diabetes Association 70^(th) Annual Scientific Sessions,    Jun. 25-29, 2010A, Orlando, Fla.-   Eldor R, Kidron M, Arbit E. Open-label study to assess the safety    and pharmacodynamics of five oral insulin formulations in healthy    subjects. Diabetes Obes Metab. March 2010B; 12(3):219-223.-   Eldor R, Kidron M, Greenberg-Shushlav Y, Arbit E. Novel    glucagon-like peptide-1 analog delivered orally reduces postprandial    glucose excursions in porcine and canine models. J Diabetes Sci    Technol. 2010C; 4(6):1516-1523.-   Kidron M, Dinh S, Menachem Y, et al. A novel per-oral insulin    formulation: proof of concept study in non-diabetic subjects. Diabet    Med. April 2004; 21(4):354-357.-   Martinez-Colubi M et al, Switching to darunavir/ritonavir    monotherapy (DRV/r mx): effect on kidney function and lipid profile.    J Int AIDS Soc. 2012 Nov. 11; 15(6):18348. doi:    10.7448/IAS.15.6.18348.-   Miyashita T et al, Hepatoprotective effect of tamoxifen on steatosis    and non-alcoholic steatohepatitis in mouse models. J Toxicol Sci.    2012; 37(5):931-42-   Ryan E A, Shandro T, Green K et al. Assessment of the severity of    hypoglycemia and glycemic lability in type 1 diabetic subjects    undergoing islet transplantation. Diabetes. 2004 April;    53(4):955-62.-   Siepmann F, Siepmann J et al, Blends of aqueous polymer dispersions    used for pellet coating: importance of the particle size. J Control    Release 2005; 105(3): 226-39.-   Sun J., Rose J. B., Bird P. (1995) J. Biol. Chem. 270, 16089-16096.-   Nissan A, Ziv E, Kidron M, et al. Intestinal absorption of low    molecular weight heparin in animals and human subjects. Haemostasis.    September-October 2000; 30(5):225-232.-   Sprecher C A, Morgenstern K A, Mathewes S, Dahlen J R, Schrader S K,    Foster D C, Kisiel W. J Biol Chem. 1995 Dec. 15; 270(50):29854-61.-   Tesauro et al. Effects of GLP-1 on Forearm Vasodilator Function and    Glucose Disposal During Hyperinsulinemia in the Metabolic Syndrome.    Diabetes Care. 2012 Oct. 15.-   Ziv E, Kidron M, Raz I, et al. Oral administration of insulin in    solid form to nondiabetic and diabetic dogs. J Pharm Sci. June 1994;    83(6):792-794.

1. An oral pharmaceutical composition, comprising an oil-based liquidformulation, said oral pharmaceutical composition comprising an insulin,a GLP-1 analogue, a trypsin inhibitor, and a chelator of divalentcations, wherein said oil-based liquid formulation is surrounded by acoating that resists degradation in the stomach.
 2. An oralpharmaceutical composition, comprising a combination of i) a firstoil-based liquid formulation, said first oil-based liquid formulationcomprising an insulin, a trypsin inhibitor, and a chelator of divalentcations; and ii) a second oil-based liquid formulation, said secondoil-based liquid formulation comprising a GLP-1 analogue, a trypsininhibitor, and a chelator of divalent cations, wherein each of saidfirst oil-based liquid formulation and said second oil-based liquidformulation is surrounded by a coating that resists degradation in thestomach.
 3. The oral pharmaceutical composition of claim 1, wherein saidoil-based liquid formulation, further comprises a component provided asa mixture of (a) a monoacylglycerol, a diacylglycerol, atriacylglycerol, or a mixture thereof; and (b) a polyethylene glycol(PEG) ester of a fatty acid.
 4. The oral pharmaceutical composition ofclaim 1, wherein said oil-based liquid formulation, further comprises aself-emulsifying component.
 5. The oral pharmaceutical composition ofclaim 1, wherein said insulin is present in an amount between 4-12 mginclusive for an adult patent or a corresponding amount per body weightfor a pediatric patient.
 6. The oral pharmaceutical composition of claim5, wherein said GLP-1 analogue is present in an amount between 100-300micrograms inclusive for an adult patent or a corresponding amount perbody weight for a pediatric patient.
 7. The oral pharmaceuticalcomposition of claim 1, wherein said GLP-1 analogue is present in anamount between 100-300 micrograms inclusive for an adult patent or acorresponding amount per body weight for a pediatric patient.
 8. Theoral pharmaceutical composition of claim 1, wherein said trypsininhibitor is selected from the group consisting of soybean trypsininhibitor (SBTI), Bowman-Birk inhibitor (BBI), and aprotinin.
 9. Theoral pharmaceutical composition of claim 1, further comprising a secondtrypsin inhibitor.
 10. The oral pharmaceutical composition of claim 9,wherein said first and second trypsin inhibitors are (i) SBTI and (ii)aprotinin; or said first and second trypsin inhibitors are (i) isolatedKTI3 and (ii) isolated BBI.
 11. The oral pharmaceutical composition ofclaim 1, wherein said chelator is EDTA.
 12. The oral pharmaceuticalcomposition of claim 1, wherein said oil is fish oil.
 13. The oralpharmaceutical composition of claim 1, wherein said oil-based liquidformulation is water-free.
 14. The oral pharmaceutical composition ofclaim 1, wherein said GLP-1 analogue is exenatide.
 15. The oralpharmaceutical composition of claim 1, wherein said coating is apH-sensitive capsule.
 16. The oral pharmaceutical composition of claim 1for reducing preprandial glucose excursion in a having human Type 2diabetes mellitus.
 17. The oral pharmaceutical composition of claim 16,wherein said insulin is present in an amount between 4-12 mg inclusive,said GLP-1 analogue is exenatide, and said exenatide is present in anamount between 100-300 micrograms inclusive.
 18. Use of the oralpharmaceutical composition of claim 1 for reducing preprandial glucoseexcursion in a having human Type 2 diabetes mellitus.
 19. A method fortreating a human with Type 2 diabetes mellitus, said method comprisingthe steps of selecting a subject diagnosed with Type 2 diabetesmellitus, and administering said subject oral pharmaceutical compositionof claim 1, thereby treating a human with Type 2 diabetes mellitus. 20.A method for treating a non-human animal with diabetes mellitus, saidmethod comprising the step of administering to said non-human animal theoral pharmaceutical composition of claim 1, thereby treating a non-humananimal with diabetes mellitus.
 21. A method for reducing preprandialglucose excursion in a human with Type 2 diabetes mellitus, said methodcomprising the steps of selecting a subject diagnosed with Type 2diabetes mellitus, and administering said subject oral pharmaceuticalcomposition of claim 1, thereby reducing preprandial glucose excursionin a human with Type 2 diabetes mellitus.
 22. A method for reducingpreprandial glucose excursion in a non-human animal with diabetesmellitus, said method comprising the step of administering to saidnon-human animal the oral pharmaceutical composition of claim 1, therebyreducing preprandial glucose excursion in a non-human animal withdiabetes mellitus.